GSM7178974: HC334,snRNAseq; Ovis aries; RNA-Seq
Nuclei were extracted from frozen anterior dorsal ventral striatal (Figure 1) tissue utilising an adapted protocol from Krishnaswami et al., 2016 33. Briefly, approximately 50-100 mg of brain tissue was transferred to a dounce homogenizer containing 1mL homogenization buffer, HB (250mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris buffer pH 8.0, 1µM DTT, 1X protease inhibitor (Sigma), 0.4 U/µL RNaseIn (ThermoFisher Scientific), 0.2 U/µL Superasein (ThermoFisher Scientific), 0.1 % Triton X-100). Tissue was homogenised using 5 strokes with the loose dounce pestle A, followed by 10-15 strokes of tight dounce pestle B. The homogenate was filtered through a 40 µm strainer into 5 mL Eppendorf tubes and centrifuged at 1000 rcf (4 oC) for 8 minutes. The supernatant was removed, and the pellet was resuspended in 250 µL of HB. A 50% - 29% iodixanol gradient (OptiPrep™ Density Gradient Medium, Sigma) was prepared to allow removal of the myelin layer. 250 µL of 50 % iodixanol was added to the nuclei-HB mixture and slowly layered on top of 500 µL of 29 % iodixanol in a new Eppendorf tube. The resultant gradient was centrifuged at 13,000 rcf (4oC) for 40 minutes. The supernatant and myelin was removed and the purified nuclei pellet was resuspended in 1 mL PBS, 1 % BSA, 0.2 U/µl RNAse inhibitor. 5 µL of resuspended nuclei was stained with 5 µL trypan blue and the quality and number of nuclei was assessed using the Countess II FL Automated Cell Counter (ThermoFisher Scientific). Single nuclei RNA libraries were prepared using the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (10x Genomics) as per manufacturer's instructions.